In recent years, considerable advances have been made in the conversion of biomass into ethanol as fuel. Most of the ethanol produced from biomass up to now is based on fermentation of corn glucose in the United States and sugarcane sucrose in Brazil. Biomass which largely consists of cellulose, hemicellulose and lignin has attracted increasing attention as an important renewable source of energy. Forestry and agricultural residues are abundant and relatively inexpensive. If this material, or at least a significant part of it, could be converted into liquid fuel, this would constitute a significant contribution to solving the problem of recycling and conservation of resources.
Ethanol can be produced from lignocellulose materials, such as wood and crop residues. The cellulose and hemicellulose components of lignocellulose can be hydrolyzed to release monosaccharides which are then fermented to ethanol. However, it has been difficult to develop an economically viable process of converting cellulosic material into fermentable sugars. The research in using lignocellulose biomass for making ethanol and other products is widely reviewed, see for example Lin and Tanaka (2006 Appl. Microbiol. Biotechnol. 69:627-642) and Saha (2003, J. Ind. Microbiol. Biotechnol. 30:279-291).
Lignocellulose is a more complex substrate than starch and sugars. One of the rate-limiting and difficult tasks is the removal of lignin. Moreover, plant tissues differ tremendously with respect to size and organization. Some plant cell types have thick cell walls and a highly lignified middle lamella separating cells from one another. These cell walls must be attacked from the luminal surface out through the secondary wall (as opposed to particles of pure cellulose, which are degraded from the outside inward). In addition to constraints imposed by the structure of cellulose itself, further limitations are imposed by diffusion and transport of the cellulolytic agent to the site of attack. Thus, prior to hydrolysis of cellulose, most woody materials are subjected to a pretreatment to make the cellulose fibers within the structures more amenable and accessible to hydrolysis.
It has been observed that fermentation of wood-derived hydrolysates to ethanol is made difficult by the presence of inhibitory substances, such as furans, organic acid and various phenolic compounds. Such inhibitory compounds are formed or released during lignin degradation and hydrolysis of complex polysaccharides in wood. The kind of toxic compounds and their concentration in lignocellulose hydrolysates depend on both the raw material and the operational conditions employed for hydrolysis. Such toxic compounds can reduce significantly the efficient utilization of sugars and production of ethanol.
A number of biological, physical, and chemical detoxification methods have been tested with hydrolysates of spruce biomass (Larsson et al., 1999, Appl. Biochem. Biotechnol. 77-79, 91-103). U.S. Pat. No. 7,067,303 discloses the use of the fungus Coniochaeta ligniaria to deplete furans in hydrolysates. One commonly used method known as “overliming” involves adjusting the pH initially to 10-11 with an alkali, e.g., calcium hydroxide or ammonia and then to 5.0-6.0 with an acid, e.g., sulfuric or phosphoric acid. The conditions used for detoxification with alkali must be carefully controlled to optimize the positive effects and minimize the degradation of fermentable sugars. The mechanisms behind the alkali detoxification effect and the influence of the choice of cation and conditions are not well understood. (Nilvebrant et al., 2003, Appl Biochem Biotechnol. 105-108:615-28; Persson et al., 2002, J Agric Food Chem. 50(19):5318-25) While overliming is quite effective, it results in an insoluble precipitate that persists through subsequent steps and must be removed and disposed of resulting in waste and increased cost.
The effectiveness of a detoxification method is variable because each type of hydrolysate has a different degree of toxicity, and each species or even strain of microorganism has a different degree of tolerance to inhibitors. Therefore, different detoxification methods cannot be strictly compared when hydrolysates from different sources and different microorganisms are used. (Mussatto and Roberto, 2004, Bioresour Technol. 93(1):1-10; Palmqvist and Hahn-Hagerdal, 2000, Bioresour Technol. 74:25-33; Palmqvist and Hahn-Hagerdal, 2000, Bioresour Technol. 74:17-24). 10010) While the focus of much of the research are directed to detoxifying wood hydrolysates for ethanolic fermentation, the use of lignocellulose biomass as feedstock for other biotechnological processes is relatively unexplored. For example, due to the increased demand for cellulase enzymes in a variety of industries, a need clearly exists for novel methods to increase cellulase production from fungi, e.g., Trichoderma reesei, such that cellulase enzymes can be more economically available.
Even within the context of ethanol generation, there is an urgent need for improvement in the economy and efficiency of the various different approaches in converting lignocellulose to sugars to ethanol.